Peroxisome proliferator-activated receptor-gamma activation inhibits tumor metastasis by antagonizing Smad3-mediated epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) was shown to confer tumor cells with abilities essential for metastasis, including migratory phenotype, invasiveness, resistance to apoptosis, evading immune surveillance, and tumor stem cell traits. Therefore, inhibition of EMT can be an important therapeutic strategy to inhibit tumor metastasis. Here, we show that activation of peroxisome proliferator-activated receptor γ (PPAR-γ) inhibits transforming growth factor β (TGF-β)-induced EMT in lung cancer cells and prevents metastasis by antagonizing Smad3 function. Activation of PPAR-γ by synthetic ligands (troglitazone and rosiglitazone) or by a constitutively active form of PPAR-γ prevents TGF-β-induced loss of E-cadherin expression and inhibits the induction of mesenchymal markers (vimentin, N-cadherin, fibronectin) and matrix metalloproteases. Consistently, activation of PPAR-γ also inhibited EMT-induced migration and invasion of lung cancer cells. Furthermore, effects of PPAR-γ ligands were attenuated by siRNA-mediated knockdown of PPAR-γ, indicating that the ligand-induced responses are PPAR-γ dependent. Selective knockdown of Smad2 and Smad3 by siRNA showed that TGF-β-induced EMT is Smad3 dependent in lung cancer cells. Activation of PPAR-γ inhibits TGF-β-induced Smad transcriptional activity but had no effect on the phosphorylation or nuclear translocation of Smads. Consistently, PPAR-γ activation prevented TGF-β-induced transcriptional repression of E-cadherin promoter and inhibited transcriptional activation of N-cadherin promoter. Finally, treatment of mice with troglitazone or knockdown of Smad3 in tumor cells significantly inhibited TGF-β-induced experimental metastasis in SCID-Beige mice. Together, with the low toxicity profile of PPAR-γ ligands, our data show that these ligands may serve as potential therapeutic agents to inhibit metastasis.

Reka AK ，Kurapati H ，Narala VR ，Bommer G ，Chen J ，Standiford TJ ，Keshamouni VG ... -  《-》

Kaempferol Suppresses Transforming Growth Factor-β1-Induced Epithelial-to-Mesenchymal Transition and Migration of A549 Lung Cancer Cells by Inhibiting Akt1-Mediated Phosphorylation of Smad3 at Threonine-179.

Kaempferol, a natural dietary flavonoid, is well known to possess chemopreventive and therapeutic anticancer efficacy; however, its antimetastatic effects have not been mechanistically studied so far in any cancer model. This study was aimed to investigate the inhibitory effect and accompanying mechanisms of kaempferol on epithelial-to-mesenchymal transition (EMT) and cell migration induced by transforming growth factor-β1 (TGF-β1). In human A549 non-small lung cancer cells, kaempferol strongly blocked the enhancement of cell migration by TGF-β1-induced EMT through recovering the loss of E-cadherin and suppressing the induction of mesenchymal markers as well as the upregulation of TGF-β1-mediated matrix metalloproteinase-2 activity. Interestingly, kaempferol reversed TGF-β1-mediated Snail induction and E-cadherin repression by weakening Smad3 binding to the Snail promoter without affecting its C-terminus phosphorylation, complex formation with Smad4, and nuclear translocation under TGF-β1 stimulation. Mechanism study revealed that the phosphorylation of Smad3 linker region induced by TGF-β1 was required for the induction of EMT and cell migration, and selective downregulation of the phosphorylation of Smad3 at Thr179 residue (not Ser204, Ser208, and Ser213) in the linker region was responsible for the inhibition by kaempferol of TGF-β1-induced EMT and cell migration. Furthermore, Akt1 was required for TGF-β1-mediated induction of EMT and cell migration and directly phosphorylated Smad3 at Thr179, and kaempferol completely abolished TGF-β1-induced Akt1 phosphorylation. In summary, kaempferol blocks TGF-β1-induced EMT and migration of lung cancer cells by inhibiting Akt1-mediated phosphorylation of Smad3 at Thr179 residue, providing the first evidence of a molecular mechanism for the anticancer effect of kaempferol.

Jo E ，Park SJ ，Choi YS ，Jeon WK ，Kim BC ... -  《NEOPLASIA》

Resveratrol suppresses epithelial-to-mesenchymal transition in colorectal cancer through TGF-β1/Smads signaling pathway mediated Snail/E-cadherin expression.

Ji Q ，Liu X ，Han Z ，Zhou L ，Sui H ，Yan L ，Jiang H ，Ren J ，Cai J ，Li Q ... -  《BMC CANCER》

TGF-β-activated SMAD3/4 complex transcriptionally upregulates N-cadherin expression in non-small cell lung cancer.

Epithelial-mesenchymal transition (EMT) is a key process in early stage of cancer metastasis. TGF-β-mediated EMT is characterized by repression of E-cadherin and induction of N-cadherin (CDH2) in various cancers. Although many investigations have focused on the regulation of E-cadherin expression, the transcription-mediated events that directly induce N-cadherin expression in TGF-β-induced EMT are not fully clear. Here, we mainly focus on non-small cell lung cancer (NSCLC) cells, in which expression of CDH2 can be activated upon TGF-β stimulation, to investigate the underlying mechanisms of CDH2 expression regulation. Western blot analysis, real-time quantitative reverse transcriptase PCR, luciferase reporter gene assays, RNA interference and in vivo chromatin immunoprecipitation (ChIP) assay were performed on human NSCLC cell lines A549 and SPC-A1. Twenty-six paired NSCLC tissues and adjacent noncancerous lung tissues were collected. Luciferase reporter assay revealed that a functional TGF-β-response element was located at position -1078 to -891 in the CDH2 promoter region. Furthermore, in vivo ChIP experiment indicated that TGF-β-activated SMAD3/4 complex was directly recruited to CDH2 promoter region (-1078 to -891). Upon TGF-β1 stimulation, knockdown of SMAD3 or/and SMAD4 led to a significant reduction in CDH2 promoter activity, and silencing of SMAD3 or SMAD4 significantly inhibited CDH2 mRNA and protein expression in A549 and SPC-A1 cells. In human NSCLC tissues, SMAD3 or SMAD4 mRNA level was positively correlated with CDH2 mRNA level, respectively. We found that TGF-β-activated SMAD3/4 complex may upregulate CDH2 expression by directly interacting with a specific SMAD-binding element in CDH2 promoter. Our findings provide insights into mechanisms underlying the transcriptional regulation of CDH2 expression in TGF-β-induced EMT and SMADs-based therapeutic strategies for NSCLCs.

Yang H ，Wang L ，Zhao J ，Chen Y ，Lei Z ，Liu X ，Xia W ，Guo L ，Zhang HT ... -  《-》

Glaucocalyxin A reverses EMT and TGF-β1-induced EMT by inhibiting TGF-β1/Smad2/3 signaling pathway in osteosarcoma.

Metastatic osteosarcoma usually has an unsatisfactory response to the current standard chemotherapy and causes poor prognosis. Currently, epithelial-mesenchymal transition (EMT) is reported as a critical event in osteosarcoma metastasis. Glaucocalyxin A, a bioactive ent-kauranoid diterpenoid, exerts anti-cancer effect on osteosarcoma by inducing apoptosis in previous study. However, the effect of Glaucocalyxin A on EMT and metastasis of osteosarcoma is unclear. In this study, we investigated the potential mechanisms of Glaucocalyxin A on EMT and metastasis of osteosarcoma. We found that Glaucocalyxin A inhibited migration and invasion of MG-63 and 143B cells. Moreover, Glaucocalyxin A increased the protein and mRNA levels of E-cadherin and decreased the protein and transcription expression of N-cadherin, Vimentin. Glaucocalyxin A also inhibited the protein and mRNA levels of EMT-associated transcription factor including Snail and Slug. Furthermore, Glaucocalyxin A inhibited transforming growth factor-β1 (TGF-β1)-induced migration, invasion and EMT of low-metastatic osteosarcoma U2OS cells. Glaucocalyxin A inhibited TGF-β-induced phosphorylation of Smad 2/3 in osteosarcoma U2OS cells. Finally, we established transplanted metastatic models of highly metastatic osteosarcoma 143B cells. Glaucocalyxin A inhibited lung metastasis in vivo. Interestingly, Glaucocalyxin A increased the protein expression of E-cadherin and reduced the protein expression of N-cadherin and Vimentin. Glaucocalyxin A inhibited the protein expression of Snail and Slug in vivo. In summary, this study demonstrated that Glaucocalyxin A inhibited EMT and TGF-β1-induced EMT by inhibiting TGF-β1/Smad2/3 signaling pathway in osteosarcoma. Therefore, Glaucocalyxin A might be a promising candidate against the metastasis of human osteosarcoma.

Jiang X ，Zhang Z ，Song C ，Deng H ，Yang R ，Zhou L ，Sun Y ，Zhang Q ... -  《-》